Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Proliferation of Reactive Astrocytes In Vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalamus. Recently, we have shown that the PACAP receptor (PAC1-R) is expressed in reactive astrocytes following an in vivo stub wound brain injury. However, the functional role of PACAP has not yet been clarified. In order to investigate the effect of PACAP on the proliferation of reactive astrocytes, a scratch wound paradigm was applied to astrocytic monolayers. Following injury, there was an increase in PAC1-R and glial fibrillary acidic protein (GFAP) immunoreactivity in the astrocytes surrounding the scratch line. PACAP at concentrations of 10^?15 to 10^?7?M was applied immediately after scratching, and the proliferating astrocytes were visualized by multiple immunofluorescence labeling. The percentage of cells that colabeled for Ki67 (a marker of proliferating cells) and GFAP increased in the 10^?11- and 10^?13-M PACAP-treated groups. The proliferating astrocytes induced by PACAP treatment mainly occurred in the proximal wound area where many reactive astrocytes were observed. Pretreatment with the PACAP receptor antagonist PACAP6-38 significantly suppressed the PACAP-induced effects. These results strongly suggest that PACAP plays an important role in the proliferation of reactive astrocytes following nerve injury.     

Mechanism of Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Human Nonfunctioning Pituitary Tumors

Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca²⁺]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca²⁺]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 ± 10 to 214 ± 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 ± 5.4 to 73.2 ± 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6–27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca²⁺]i rise in cells obtained from 7 NFPA (from 110 ± 34 to 151 ± 40 nM [Ca²⁺]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 μM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca²⁺ as well as blockade of L-type Ca²⁺ channels by dihydropyridine antagonists abolished [Ca²⁺]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.     

Pituitary Adenylate Cyclase-Activating Polypeptide Deficiency Enhances Oxazolone-Induced Allergic Contact Dermatitis in Mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in capsaicin-sensitive sensory neurons and inflammatory/immune cells, therefore it is suggested to play a role in neuro-immune interactions. Our aim was to investigate the role of PACAP in oxazolone-induced delayed-type hypersensitivity reaction in the skin using deficient mice (PACAP^-/-). Sensitization was induced by 2% oxazolone application on the shaved abdomen on two consecutive days; inflammation was elicited by oxazolone smearing on the ears 6 days later. Ear thickness was measured by micrometry. Histological examination, cytokine profile [IL-2, IL-4, IL-5, and monocyte chemoattractant protein-1: MCP-1, IFN-γ, tumor necrosis factor alpha (TNF-α)] and myeloperoxidase activity correlating with the number of neutrophils/macrophages were determined 24 and 48 h later. Oxazolone induced a 110–130% swelling after 24–48 h in wild-type mice, which was significantly greater in PACAP-deficient mice. Histological analysis confirmed markedly increased edema in PACAP^-/- mice, but the moderately enhanced inflammatory cell accumulation was not statistically significant compared with the wild-types. There was no difference in myeloperoxidase activity of the ear homogenates. Elevation of MCP-1, but not the levels of the other cytokines, was significantly higher in the samples of the PACAP-deficient mice. These results suggest that PACAP exerts anti-inflammatory, particularly edema-inhibiting effects in allergic contact dermatitis.     

Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) onHormone Secretion from Sheep Pituitary Cells in vitro

Although vasoactive intestinal polypeptide (VIP) is thought to be a prolactin releasing factor, in vivo studies on sheep suggest that it is inactive in this species. Recent studies, based primarily on the rat, suggest that the related pituitary adenylate cyclase-activating polypeptide (PACAP) is also a hypophysiotrophic factor but again in sheep, this peptide has no in vivo effects on hormone secretion despite being a potent activator of adenylate cyclase in vitro. This lack of response to either peptide in vivo in sheep could be due to the low concentration of peptide that reaches the pituitary gland following peripheral injection. In the present study we therefore adopted an alternative approach of evaluating in vitro effects of these peptides on GH, FSH, LH or prolactin secretion from dispersed sheep pituitary cells. In a time-course study, PACAP (1 μmol/l) increased GH concentrations in the culture medium between 1 and 4 h and again at 12 h but had no effect in the 6 and 24 h incubations. Prolactin, LH and FSH were not affected by PACAP. The response to various concentrations of PACAP (1 nmol/l–1 μmol/l) were then evaluated using a 3 h incubation. Again prolactin and LH were not affected by PACAP and there was a small increase in GH concentrations but only at high concentrations of PACAP (0.1 and 1 μmol/l; P<0.05). PACAP also stimulated FSH secretion in cells from some animals although this effect was small. The GH response to PACAP was inhibited by PACAP(6–38), a putative PACAP antagonist, but not by (N-Ac-Tyr¹, D-Arg²)-GHRH(1–29)-NH_2, a GH-releasing hormone (GHRH) antagonist. The cAMP antagonist Rp-cAMPS was unable to block the GH response to PACAP suggesting that cAMP does not mediate the secretory response to this peptide. At incubation times from 1–24 h, VIP (1 μmol/l) had no effects on prolactin, LH or GH secretion and, in a further experiment based on a 3 h incubation, concentrations of VIP from 1 nmol/l–1 μmol/l were again without effect on prolactin concentrations. Interactions between PACAP and gonadotrophin releasing hormone (GnRH), GHRH and dopamine were also investigated. PACAP (1 nmol/l–1 μmol/l) did not affect the gonadotrophin or prolactin responses to GnRH or dopamine respectively. However, at a high concentration (1 μmol/l), PACAP inhibited the GH response to GHRH. In summary, these results show that PACAP causes a modest increase in FSH and GH secretion from sheep pituitary cells but only at concentrations of PACAP that are unlikely to be in the physiological range. The present study confirms that VIP is not a prolactin releasing factor in sheep.     

Functional Characterization of Neural-Restrictive Silencer Element in Mouse Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Gene Expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) is predominantly localized in the nervous system, but the underlying mechanism in its neuron-specific expression remains unclear. In addition to two neural-restrictive silencer-like element (NRSLE1 and 2), as reported previously, we have identified the third element in ?1,601 to ?1,581 bp from the translational initiation site of mouse PACAP gene and termed it as NRSLE3, of which, the sequence and location were highly conserved among mouse, rat, and human PACAP genes. In luciferase reporter assay, the deletion or site-directed mutagenesis of NRSLE3 in the reporter gene construct, driven by heterologous SV40 promoter, cancelled the repression of luciferase activity in non-neuronal Swiss-3T3 cells. Furthermore, its promoter activity was significantly repressed in Swiss-3T3 cells, but not in neuronal-differentiated PC12 cells. The electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells demonstrated a specific complex with NRSLE3 probe that exhibited the same migration with the neural-restrictive silencer element (NRSE) probe of rat type II sodium channel gene. During neuronal differentiation of PC12 cells, the increment of PACAP mRNA exhibited the correlation with that of REST4 mRNA, which is a neuron-specific variant form of neural-restrictive silencer factor (NRSF). In undifferentiated PC12 cells, trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which indirectly inhibits NRSF-mediated gene silencing, increased PACAP mRNA level and attenuated the repression of promoter activity of 5′ flanking region of mouse PACAP gene containing NRSLEs. These suggest that the NRSE–NRSF system implicates in the regulatory mechanism of neuron-specific expression of PACAP gene.     

Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs—neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H₂O₂ for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H₂O₂ and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.     

Pituitary Adenylate Cyclase-Activating Polypeptide Prevents Cisplatin-Induced Renal Failure

Cisplatin is widely used for cancer chemotherapy, but nephrotoxicity is a major dose-limiting side effect. Our recent studies in vitro have shown that pituitary adenylate cyclase-activating polypeptide (PACAP) ameliorated cisplatin nephrotoxicity and that the renoprotection with PACAP38 was mediated by the PAC₁ receptor and through the p53-dependent and -independent suppression of apoptosis of human renal proximal tubular epithelial cells. In the present studies, PACAP38 prevented the rise in blood urea nitrogen and serum creatinine in mice treated with cisplatin. Cisplatin-exposed mice treated with PACAP38 had relatively well-preserved tubular integrity, even when the treatment started 24 h after cisplatin exposure. PACAP38 also reduced plasma and kidney levels of tumor necrosis factor-α and restored collagen IV levels. The damage to mouse kidney tubules caused by cisplatin involved p53 accumulation and was partially reversed by treatment with PACAP38. PACAP38 ameliorates cisplatin-induced acute kidney injury even when treatment started 24 h after the onset of injury and increases tubular regeneration, which further facilitates restoration of kidney function in addition to its anti-apoptotic effects.     

Comparison of Expression and Proliferative Effect of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its Receptors on Human Astrocytoma Cell Lines

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide considered to be a potent regulator of astrocytes. It has been reported that PACAP also affects astrocytoma cell properties, but the proliferative effects of this peptide in previous reports were inconsistent. The purpose of this study was to search for correlations between malignant potential, PACAP/PACAP receptor expression, and the proliferative potential of four astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13). Immunohistochemical observations were performed using astrocyte lineage markers with a view to establishing malignant potential, which is inversely correlated to differentiation status in astrocytoma cells. YH-13 showed the most undifferentiated astrocyte-like status, and was immunopositive to a cancer stem cell marker, CD44. These observations suggest that YH-13 is the most malignant of the astrocytoma cell lines tested. Moreover, the strongest PAC1-R immunoreactivity was observed in YH-13 cells. Using real-time PCR analysis, no significant differences among cell lines were detected with respect to PACAP mRNA, but PAC1-R and VPAC1-R mRNA levels were significantly increased in YH-13 cells compared with the other cell lines. Furthermore, when cell lines were treated with PACAP (10^?11 M) for 3 days, the YH-13 cell line, but not of the other cell lines, exhibited a significantly increased cell number. These results suggest that PACAP receptor expression is correlated with the malignant and proliferative potential of astrocytoma cell lines.     

Implication of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) for Neuroprotection of Nicotinic Acetylcholine Receptor Signaling in PC12 Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neurotrophic factor through PAC1-R, PACAP-specific receptor, in the central nervous system. On the other hand, by interacting with nicotinic acetylcholine receptor (nAChR), nicotine exhibits several neuroprotective effects. Since the relevance of PACAP and nAChR signaling has not been reported so far, we attempted to investigate their relevance in terms of neuroprotection in PC12 cells. Regarding the effect of nicotine on PACAP gene expression, nicotine increased its mRNA level in time-dependent and dose-dependent manners in the PC12 cells differentiated with nerve growth factor (NGF). In addition, luciferase reporter assay demonstrated that nicotine treatment significantly augments the promoter activity of PACAP gene. Since PAC1-R mRNA expression was induced by NGF, PACAP exhibited neuroprotective effect against tunicamycin-induced cell death in the differentiated PC12 cells. Nicotine also exhibited the neuroprotective effect, which was significantly attenuated by Max.d.4 (PAC1-R specific antagonist). These results suggest that the increment of PACAP gene expression due to nicotine treatment might be involved in the neuroprotection by nicotine.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Actions on αT3-1 Gonadotrophs Show Desensitization

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived αT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation. In acute experiments, PACAP27-stimulated cAMP accumulation (intracellular plus extracellular) was measured (in the presence of phosphodiesterase inhibitor) both in intact cells and in cell membranes. The peptide increased cAMP accumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in both cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (although the deviation from linearity was less pronounced than with PACAP27). Thus, rapid homologous desensitization to PACAP27 occurs in intact αT3-1 cells, but is not entirely receptor specific. Rapid homologous desensitization of PACAP27-stimulated cAMP accumulation also occurred in the presence of a protein kinase C activating phorbol ester, which inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to cause a spike-type elevation of cytosolic Ca²⁺ concentration (a consequence of phosphoinositidase C activation). In chronic desensitization studies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC₅₀ approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%). Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) adenylyl cyclase undergoes rapid homologous desensitization in αT3-1 cells, which is paralleled by homologous desensitization of PACAP27-stimulated phosphoinositidase C activity and involves mechanisms distinct from those underlying heterologous desensitization by phorbol esters. Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.     

Signalling Pathways Mediating Secretory and Mitogenic Responses to Galanin and Pituitary Adenylate Cyclase-Activating Polypeptide in the 235-1 Clonal Rat Lactotroph Cell Line

The neuropeptides galanin and pituitary adenylate cyclase-activating peptide (PACAP) have been implicated in the physiological regulation of lactotroph function. Using the 235-1 clonal lactotroph rat cell line we have studied the signalling pathways mediating the secretory and mitogenic responses to galanin and PACAP. Both peptides stimulated prolactin release to a similar maximal extent. PACAP (100  nM) stimulated an increase in the proliferation rate of 235-1 cells, but was significantly less effective than 100  nM galanin (161.8±2.3% vs 296.1±9.1% of control). PACAP stimulated cAMP accumulation with an ED₅₀ of 3.2  nM, and a maximal effect of almost two-fold at a concentration of 100  nM. Galanin depleted cAMP, by 30% at a concentration of 100  nM. The aminosteroid phospholipase C (PLC) inhibitor U-73122 virtually abolished maximal peptide stimulated prolactin release. Depletion of inositol phosphates or downregulation of protein kinase C reduced maximal peptide stimulated prolactin release from about 260% to about 160% of unstimulated release. Both peptides at a concentration of 100  nM caused a sustained increase in intracellular calcium when incubated with cells for 30  min. These results demonstrate that both peptides stimulate prolactin release and the proliferation rate of 235-1 cells. The most important signalling pathway for prolactin release activated by both peptides is via PLC, although they also regulate cAMP levels, which are increased by PACAP and decreased by galanin. Despite maximal peptide stimulated prolactin release being equal, galanin has a greater mitogenic effect on 235-1 cells than PACAP, raising the possibility that it activates an additional mitogenic signalling pathway.     

Correlation Between Oocyte Number and Follicular Fluid Concentration of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Women After Superovulation Treatment

Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormone-signaling cascades in the ovary. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n?=?132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290?fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290?fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, high-oocyte number, and low-PACAP concentration-low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0?fmol/ml, respectively, while the median values of harvested oocytes were 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Induces Relaxations of Peripheral and Cerebral Arteries,which are Differentially Impaired by Aging

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a well-known neuropeptide, which also has vasomotor effects. However, little is known regarding its age-related and organ-specific vasomotor effects. We hypothesized that the vasomotor effects of PACAP depend on the tissue origin of the vessels and aging substantially modulates its actions. Thus, carotid (CA) and basilar arteries (BA) were isolated from young (2 months old), middle age (12 months old), and old (30 months old) rats. Their vasomotor responses were measured with an isometric myograph (DMT610M) in response to cumulative concentrations of PACAP1-38 (10^?9–10^?6 M). PACAP1-38 induced (1) significantly greater concentration-dependent relaxations in CA compared to that of BA of young, middle age, and old rats; (2) relaxations of CA significantly decreased, whereas they did not change substantially in BA, as a function of age; (3) sodium nitroprusside (SNP)-induced relaxation did not change after PACAP1-38 administration in any conditions; and (4) inhibition of PAC1 receptors by selective PAC1 receptor blocker (PACAP6-38) completely diminished the responses to PACAP in all age groups of BA and CA. In conclusion, these findings suggest that PACAP1-38 has greater vasomotor effect in CA than that in BA, whereas aging has less effect on PACAP-induced relaxation of cerebral arteries and BA than that in peripheral arteries and CA suggesting that the relaxation to PACAP is maintained in cerebral arteries even in old age.     

Localization of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in the Hypothalamus-Pituitary System in Rats: Light and Electron Microscopic Immunocytochemical Studies

The localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the hypothalamus-pituitary system in rats was examined in light and electron microscopic immunocytochemistry using a specific antiserum to synthetic PACAP 1–38 (R0831). In light microscopic study, intensely PACAP-immunostained perikarya were observed in the supraoptic and paraventricular magnocellular nucleus in the hypothalamus. In the median eminence, many immunoreactive nerve fibers were observed in the internal layer, but a few immunoreactive terminals were noticed in the external layer. In the pituitary gland, numerous immunoreactive nerve fibers were observed in the posterior lobe. In the intermediate lobe, moderately immunostained cells were observed, but in the anterior lobe no immunostained cells were noticed. In electron microscopic study, PACAP-immunoreactivity was examined by avidin-biotin peroxidase complex method. In the perikarya of the supraoptic and paraventricular magnocellular nucleus, DAB-reaction products were distributed diffusely in the cytoplasmic matrix, frequently attaching to the rough-surfaced endoplasmic reticulum. In the nerve terminals of the posterior lobe, reaction products were observed among the secretory granules, but sometimes upon them. In the cells of the intermediate lobe, reaction products were also distributed in the cytoplasmic matrix.     

Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Vasoactive Intestinal Polypeptide (VIP) on Prolactin, Luteinizing Hormone and Growth Hormone Secretion in the Ewe

This study was undertaken to investigate the roles of PACAP and VIP in the control of pituitary hormone secretion in the ewe. The first experiment was designed to identify any direct effects at the level of the pituitary and was conducted during the luteal phase of a prostaglandin-synchronized oestrous cycle. PACAP (0.008, 0.04, 0.2 and 1.0 nmol/min) or VIP (0.06, 0.2, 0.6 and 1.8 nmol/min) was infused into the carotid artery over a 10 min period. Blood samples were taken before and after the infusions so that plasma PRL, LH and GH concentrations could be measured. Blood pressure was also monitored to determine if the doses used were biologically active. In no case was an effect on hormone secretion observed. In contrast, the highest dose of each peptide induced an increase in heart rate to almost three-fold the resting value. Although both peptides are active in vivo, this result suggests that neither peptide has a direct effect on hormone release from the pituitary of prostaglandin-synchronized ewes. In a second experiment, we investigated whether the peptides had central effects on hormone secretion. Intracerebroventricular (ICV) injection of PACAP or VIP at the dose 10nmol was tested in ovariectomized ewes. After injection, PACAP suppressed PRL and GH secretion so that plasma hormone concentrations from 1–3 h after injection were significantly different from the control (P<0.05 for PRL, P<0.01 for GH). In addition, PACAP significantly reduced mean LH concentration (P<0.05) and LH pulse frequency (P<0.01). A similar suppressive effect on LH secretion was also observed after ICV injection of VIP (P<0.05 for both parameters), although PRL and GH release were not affected. These results suggest a possible role for PACAP in the neuroendocrine control of PRL, GH and LH secretion in sheep. In addition, VIP may be involved in the control of LH secretion. In contrast, there is no evidence to suggest that either peptide is a hypophysiotropic factor for PRL, LH or GH in prostaglandin-synchronized ewes.     

Characterization of the Thermoregulatory Response to Pituitary Adenylate Cyclase-Activating Polypeptide in Rodents

Administration of the long form (38 amino acids) of pituitary adenylate cyclase-activating polypeptide (PACAP38) into the central nervous system causes hyperthermia, suggesting that PACAP38 plays a role in the regulation of deep body temperature (T _b). In this study, we investigated the thermoregulatory role of PACAP38 in details. First, we infused PACAP38 intracerebroventricularly to rats and measured their T _b and autonomic thermoeffector responses. We found that central PACAP38 infusion caused dose-dependent hyperthermia, which was brought about by increased thermogenesis and tail skin vasoconstriction. Compared to intracerebroventricular administration, systemic (intravenous) infusion of the same dose of PACAP38 caused significantly smaller hyperthermia, indicating a central site of action. We then investigated the thermoregulatory phenotype of mice lacking the Pacap gene (Pacap ^?/?). Freely moving Pacap ^?/? mice had higher locomotor activity throughout the day and elevated deep T _b during the light phase. When the Pacap ^?/? mice were loosely restrained, their metabolic rate and T _b were lower compared to their wild-type littermates. We conclude that PACAP38 causes hyperthermia via activation of the autonomic cold-defense thermoeffectors through central targets. Pacap ^?/? mice express hyperkinesis, which is presumably a compensatory mechanism, because under restrained conditions, these mice are hypometabolic and hypothermic compared to controls.